Chromatography is a method of chemical analysis based upon the principles of phase distribution that uses differences in a variety of molecular properties to separate chemical compounds. Chromatography systems consist of a mobile phase and a stationary phase, where components of the sample to be separated are carried with varying migration rates by the mobile phase as it flows through the stationary phase. The more securely a component of the sample is held by the stationary phase, the higher the percentage of molecules of that component that will be held immobile. In contrast, a less securely held component will provide a higher percentage of molecules moving with the mobile phase. On average, the molecules of a less securely held component will move over the stationary phase, in the direction of flow, at a higher rate than the molecules of a more securely held component. As a result, components in the sample will tend to migrate into separate regions, or bands, of the stationary phase.
A number of different types of chromatography are well known including column chromatography thin-layer chromatography, gas chromatography and paper chromatography. Thin-layer chromatography (TLC), is characterized by a liquid mobile phase and a thin stationary phase which is supported by a rigid glass, plastic, or metal plate. TLC is performed by depositing a sample, which is dissolved in a volatile solvent, at the bottom of a TLC plate using a pipet or syringe in either a spot or line application. The solvent evaporates in the process, leaving the sample on the stationary phase. The plate is then subjected to a development process which separates components of the sample for identification.
For proper results, care is required in depositing samples on the TLC plate. The plate itself must be held substantially horizontally to prevent run-off of samples deposited thereon. Additionally, the syringe or pipet used for depositing the sample is typically held at an angle between zero and 45 degrees for optimum accuracy.
More importantly, however, the size of the sample deposited on the chromatography plate is limited by the type and thickness of the stationary phase coated on the plate. This is particularly critical in spot application of samples. In line application, the sample must also be deposited evenly. Line application of a sample is typically accomplished by manually moving the syringe or pipet linearly in a direction substantially perpendicular to the direction of mobile phase flow desired for the sample. In both spot and line applications, resolution is determined primarily by the size of the sample applied. To obtain the best resolution with TLC, the sample is typically applied as a small spot of 1 mm or 2 mm diameter or in a thin line of similar width. This is usually accomplished by applying several small spots on top of each other or in a line. Moreover, in the process, the solvent must be allowed to completely evaporate before additional sample spots are applied at a given location.
As a result, an experienced technician is typically required to effect the application of samples to a TLC plate. Moreover, even an experienced technician can generally perform only one application at a time. The number of applications that can be performed in a given time is therefore limited.
A number of mechanical devices are known for fluid application generally. U.S. Pat. Nos. 2,764,980 issued to Smith, 2,786,468 issued to Singer et al and 4,846,797 issued to Howson et al disclose machines for injecting small quantities of liquid into living organisms. The machines include multiple housings and syringes. Multiple syringe plunger actuators can be driven either simultaneously or independently.
U.S. Pat. No. 4,288,206 issued to Tigwell et al discloses an automatic multiple water sampler including a stationary housing adapted to receive multiple syringes and a cam for simultaneously engaging or disengaging the syringe plungers. U.S. Pat. No. 4,931,041 issued to Faeser discloses an infusion syringe pump including a syringe housing adapted to receive a single syringe. A linearly moveable drive member actuates the syringe plunger and is also part of a control system used to determine the absolute plunger position.
The above noted devices, however, are not designed for fluid application in TLC. Specifically, these devices are not designed for use in conjunction with a TLC plate and therefore are unable to accurately apply samples to such a plate. This is particularly true with respect to line application.
U.S. Pat. No. 4,407,659 issued to Adam discloses a drive system for depositing samples for chemical analysis. The system includes a single syringe housing adapted to receive a syringe and a coupling block adapted to engage or retract the syringe plunger. The device, however, is not specifically designed for accurately depositing samples onto a TLC plate. Significantly, the device lacks the ability to effect line application of samples and does not overcome the problem of a limited number of samples that may be applied in a given time.
U.S. Pat. No. 5,055,271 issued to Golias et al discloses a pump inserter for test tubes. The device is relevant to the extent that it discloses a vertically moveable frame for inserting and retracting pump mechanisms into closed test tubes and a horizontally moveable frame for positioning the test tubes beneath the pump mechanisms.
There are also several commercial products designed to mechanically augment the process of depositing samples on a TLC plate. Most such systems utilize only a single syringe or pipet and therefore do not overcome the problem of a limited number of samples that can be applied in a given time. Moreover, none of these systems are capable of effecting both line and spot applications with multiple syringes or pipettes.
Other automated systems for depositing samples on a TLC plate spot fluid from a plurality of syringes but provide no mechanism for line application. Such systems also have no provisions for automatically cleaning the syringes and, if a single syringe happens to jam, the plating of the remaining syringes will cease until the jammed syringe is removed. These systems also are not designed to precisely deliver small volumes necessary for high performance analytical TLC.
The apparatus of the present invention has the control and accuracy of the best commercially available spotting units in a multiple syringe platform and allows for both line and spot application. The apparatus further allows for easy syringe cleaning after use, has special provision for syringe jamming, has a simple manual override mechanism, and is relatively inexpensive to produce. A wide range of sample volumes can be plated in spot or line geometry with a wide variety of syringes.